PROJECT SUMMARY Induced pluripotent stem cell-derived cardiomyocytes (iPS-CM) are emerging as an invaluable in vitro human experimental platform for disease modeling, drug discovery, cardiotoxicity screening, gene editing and functional genomics. For the first time, cardiac electrophysiology has access to a scalable human experimental model, which, currently, offers the only path to personalized (cardiac) medicine as patient-derived iPS-CMs can be generated on a progressively faster time scale. The clear potential of this technology motivates efforts to address the main criticisms facing iPS-CMs, namely the need for further maturation and reduction of phenotype heterogeneity. As multiple approaches are being pursued to improve iPS-CM maturation and to approximate the functionality of the adult human myocardium, we argue that combinatorial optimizations necessitate new high-throughput (HT) technology and automation. The overall goal of this project is to develop and validate a scalable platform for optimizing cardiac tissue engineering via chronic reconfigurable optical pacing and ?on-demand? oxygenation for gaining mechanistic insights into cardiac metabolism and electrophysiology, a platform we call ChROME. Chronic electrical stimulation is a viable lead to iPS-CM maturation, yet it has remained under-explored, specifically as related to the role of mass transport and oxygenation during such stimulation. Leveraging our expertise in the theoretical and experimental use of optogenetic tools for cardiac applications (Entcheva) and automation (Kostov, Li, Entcheva, Kay), we propose to design and validate the first-generation HT-ChROME platform, that will integrate continuous monitoring of key physiological parameters. Our team?s expertise in optical oxygen sensing (Kostov), in-house manufacturing of ?on-demand? oxygenation nanocarriers (perfluorocarbons, PFC) (Kay) and metabolic characterization (Kay, Beard) will be applied to address the increased metabolic demands during stimulation. The ability to quantify ?functional maturation? by relevant measures (voltage, calcium, contraction) in a high-throughput manner (Entcheva) in 2D and 3D cardiac tissue constructs (Vunjak-Novakovic), using our automated platform OptoDyCE (all-optical dynamic cardiac electrophysiology) is critical in this undertaking. Employing these HT tools and other imaging and omics modalities (Popratiloff, Horvath), we will elucidate the spectrum of responses triggered by chronic stimulation of iPS-CMs: beneficial/maturation effects vs. pathological overload effects, depending on load and oxygenation conditions. The proposed HT-ChROME platform represents a critical step in resolving issues impeding progress with iPS-CMs to accelerate their wide-spread adoption in basic and translational applications. The obtained large-scale data will inform a new generation of biophysical models linking human cardiac metabolism and electrophysiology.